Noninvasive in vivo imaging of diabetes-induced renal oxidative stress and response to therapy using hyperpolarized 13C dehydroascorbate magnetic resonance

Abstract

Oxidative stress has been proposed to be a unifying cause for diabetic nephropathy and a target for novel therapies. Here we apply a new endogenous reduction-oxidation (redox) sensor, hyperpolarized (HP) (13)C dehydroascorbate (DHA), in conjunction with MRI to noninvasively interrogate the renal redox capacity in a mouse diabetes model. The diabetic mice demonstrate an early decrease in renal redox capacity, as shown by the lower in vivo HP (13)C DHA reduction to the antioxidant vitamin C (VitC), prior to histological evidence of nephropathy. This correlates with lower tissue reduced glutathione (GSH) concentration and higher NADPH oxidase 4 (Nox4) expression, consistent with increased superoxide generation and oxidative stress. ACE inhibition restores the HP (13)C DHA reduction to VitC with concomitant normalization of GSH concentration and Nox4 expression in diabetic mice. HP (13)C DHA enables rapid in vivo assessment of altered redox capacity in diabetic renal injury and after successful treatment.

Figure 1

Schematic representation of HP DHA uptake and its subsequent reduction to VitC. The green box indicates GLUT1. DHA is reduced to VitC by DHA reductase (EC 1.8.5.1) in a glutathione-dependent mechanism via coupled reaction to NADPH.

Figure 2

Comparison of HP ¹³C DHA reduction in db/m and db/db mice. A: Representative HP ¹³C MR spectra in a 12-week db/m and db/db mouse. Kidney voxels demonstrate an approximately 50% decrease in VitC production in db/db compared with db/m mouse. B: Renal VitC/(VitC + DHA) ratios in db/db and db/m mice at 8, 12, and 16 weeks of age. *Significant difference when compared with age-matched db/m mice. #Significant difference when compared with 8-week-old db/db mice.

Figure 3

Glutathione and Nox4 in db/db and db/m mice. A: GSH and GSSG concentrations and GSH/GSSG ratios in the kidneys of the db/m and db/db mice at 8, 12, and 16 weeks of age. The GSH concentrations and GSH/GSSG ratios were significantly lower in db/db mice compared with db/m mice at all three time points. B: Renal Nox4 expression in db/m and db/db mice at 8, 12, and 16 weeks of age, demonstrating significantly higher Nox4 expression in the db/db mice compared with the db/m mice. *P < 0.05.

Figure 4

HP ¹³C DHA reduction and correlative tissue studies in db/db mice after ramipril treatment. A: Representative HP ¹³C MR spectra of a 12-week-old db/m mouse, an untreated db/db mouse, and a db/db mouse after 4 weeks of ramipril treatment. Kidney voxels demonstrate normalization of VitC production in the db/db mouse after treatment to a level similar to that seen in the db/m mouse. Renal VitC/(VitC + DHA) ratios (B); renal GSH, GSSG concentrations, and GSH/GSSG ratios (C); and renal Nox4 expressions (D) demonstrate similar findings. E: Representative immunostains of 8-OHdG in renal slices from 12-week-old db/m mice, untreated db/db mice, and treated db/db mice demonstrate increased staining, indicating increased oxidative DNA damage, in the untreated db/db mice, which is diminished with ramipril treatment. F: Twenty-four–hour urine 8-OHdG concentrations are significantly higher in the untreated db/db mice. Ramipril-treated db/db mice show urine 8-OHdG concentrations similar to those in the db/m mice. G: Representative PAS stains of renal slices from 12-week-old db/m mice (top left), untreated db/db mice (top middle), and treated db/db mice (top right), and the percent mesangial matrix area demonstrate increased mesangial matrix area in the untreated db/db mice (arrows), which is diminished with ramipril treatment. *P < 0.05.

Figure 5

Schematics illustrating the relationships among HP DHA, associated redox pairs, and Nox4. A: In response to persistent hyperglycemia, renal Nox4 is upregulated and generates ROS (superoxide) by using NADPH as an electron donor. There is decreased regeneration of GSH from GSSG and decreased reduction of HP DHA to VitC. B: Ramipril decreases Ang II production, which in turn decreases Nox4 expression and ROS generation. Via the redox pairs of VitC and GSH, the treatment effect is observed as the normalization of HP DHA reduction to VitC. The green box indicates GLUT1.